INTRODUCTION: While the rate limiting enzymatic step in steroidogenesis is the conversion of cholesterol to pregenenolone by the CSCC complex located in the mitochondria, the true rate determining step in this process is the delivery of substrate to the inner mitochondrial membrane where cleavage occurs. This process requires the synthesis of new proteins which are thought to be indispensable for cholesterol transfer. The search for this regulatory protein(s) has spanned greater than two decades and has not yet yielded unequivocal information on this problem. Using radioisotope labelling techniques followed by 2-D PAGE, we have described several mitochondrial proteins in hormone stimulated MA-10 mouse tumor Leydig cells which may be candidates for the regulatory protein(s). Earlier findings from our laboratory greatly strengthened the probability that these mitochondrial proteins were involved in acute steroidogenic regulation. In this proposal we add further evidence of the importance of these proteins and describe a model whereby they may function in the transfer of cholesterol to the inner mitochondrial membrane. Therefore, we propose to continue to pursue the purification and characterization of these proteins since they would appear to be extremely strong candidates for playing a role in steroidogenesis. SPECIFIC AIMS: Specifically, it is proposed: 1) to further characterize the synthesis, degradation, phosphorylation and protein processing profiles of mitochondrial proteins in MA-10 and R2C cells using two-dimensional polyacrylamide gel electrophoresis. 2) to use FPLC technology to purify the mitochondrial 30 kDa proteins from stimulated MA-10 cells. 3) to utilize the purified protein(s) in reconstruction experiments with isolated mitochondria in order to determine if they play a role in the acute regulation of CSCC activity. 4) to utilize the purified protein(s) for microsequencing and for the generation of polyclonal antibodies which will then be used in further characterization of the protein(s) with regards to function, rates of synthesis and localization. 5) to clone and sequence the gene for these proteins. METHODS: Mitochondrial proteins and phosphoproteins will be studied by radiolabeling, 2-D PAGE and fluorography. Quantitation of proteins will be performed using the Visage 2000 image analysis system. Steroid production will be measured by RIA. Protein purification will be accomplished using FPLC. Antibodies will be generated using standard polyclonal antibody production methods. Immunocytochemistry will be performed using the protein-A gold technique. Microsequencing of proteins will be performed by a commercial source. Cloning and sequencing of the gene will be performed using standard methodology.